1. p63 Regulates Olfactory Stem Cell Self-Renewal and Differentiation
Russell B. Fletcher, Melanie S. Prasol, Jose Estrada, Ariane Baudhuin, Karen Vranizan, Yoon Gi Choi, John Ngai 
Neuron 
Volume 72, Issue 5, Pages (December 2011)
DOI: /j.neuron
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1
p63 Is Expressed in Horizontal Basal Cells of the Olfactory Epithelium
(A) Schematic of cell types and lineage relationships in the postnatal olfactory epithelium. Multipotent horizontal basal cells (HBCs) and globose basal cells (GBCs) give rise to immature and mature olfactory sensory neurons (iOSNs and mOSNs, respectively), sustentacular support cells (SUSs), and cells of the Bowman's gland (BGs). HBCs reside directly adjacent to the basal lamina, indicated by the dashed line; apical and basal aspects of the epithelium are indicated.
(B) ICAM1(+) (enriched in HBCs; lower right quadrant) and ICAM1(−)(lower left quadrant) cells of the olfactory epithelium were purified by FACS using an FITC-labeled antibody to ICAM1. Gene expression in these purified populations of cells was assayed using Affymetrix DNA microarrays.
(C) Differential gene expression between ICAM1(+) and ICAM1(−) cells is shown in this volcano plot, which plots for each probe set the average M value (where M is the log2 ratio of expression level in ICAM1(+) versus ICAM1(−) cells, i.e., M value = log2[ICAM1(+)/ICAM1(−)]) versus −log10[p value]. Probe sets corresponding to transcripts showing high levels of enrichment and low variance reside in the upper right quadrant of the graph. Through this analysis, the transcription factor p63 was identified as one of the most highly enriched transcripts in ICAM1-positive cells (blue circle).
(D and E) RNA in situ hybridization (D) and immunohistochemistry (E) showing p63 localization to the HBC layer in postnatal (P30) olfactory epithelium. Note colocalization of p63 (nuclear) and ICAM1 (plasma membrane) in (E). Location of basal lamina in (D) and (E) is indicated by dashed line.
(F) RT-PCR analysis of p63 isoforms expressed in ICAM1(+) HBCs. ΔNp63 is the predominant N-terminal isoform; TAp63 is undetectable in this assay, as well as by qRT-PCR. All 3′ splice forms (alpha, beta, and gamma) are expressed in these cells. Scale bar represents 50 μm. See also Figure S1.
Neuron  , DOI: ( /j.neuron )
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 2
Expression of p63 during Injury-Induced Regeneration of the Olfactory Epithelium
Four-week-old mice were injected with methimazole to stimulate degeneration of differentiated olfactory epithelium cell types, followed by their regeneration from HBCs. Tissue sections from uninjured (UI) and injured olfactory epithelium at 1, 2, 3, 5, and 7 days post-injury (dpi) were analyzed by double-label immunohistochemistry and confocal microscopy.
(A–F) HBCs and their descendants were labeled with an anti-GFP/YFP antibody in olfactory epithelium from Krt5CrePR;Rosa26YFP mice, and proliferative cells were detected using an antibody against Ki67.
(G–L) Immunohistochemical localization of p63 and Ki67. Note that p63 is expressed exclusively by HBCs in the uninjured epithelium, where they are mostly quiescent. Upon injury, HBCs proliferate and differentiate to regenerate the pseudostratified olfactory epithelium, as evidenced by the progressive addition of suprabasally localized YFP-labeled cells. p63 expression remains localized to the HBC layer during injury-induced regeneration and is expressed transiently in proliferating HBCs, whose numbers peak at 2 dpi. Locations of basal lamina are indicated by dashed lines; apical and basal aspects of the epithelium are indicated in (C). Images were captured from the septum in the middle and ventral regions of the olfactory epithelium. Scale bar represents 50 μm. See also Figure S2.
Neuron  , DOI: ( /j.neuron )
Copyright © 2011 Elsevier Inc. Terms and Conditions

4. Figure 3
p63 Is Required for Maintenance of HBC Cell Fate, but Not for HBC Differentiation, during Injury-Induced Regeneration
Olfactory epithelium from Krt5-CrePR;Rosa26YFP mice in the p63+/ and p63lox/lox backgrounds was analyzed by immunohistochemistry 6 days following methimazole-induced injury at P16. Tissue sections were colabeled with anti-GFP/YFP to localize HBCs and their descendants, as well as various markers of specific olfactory epithelium cell types, and they were visualized by confocal microscopy. By 6 days post-injury in control p63+/ tissue, HBCs have regenerated the multilayered, pseudostratified epithelium that includes cells expressing p63 (A), Krt14 (B), and ICAM1 (C) markers for HBCs, the proliferative marker Ki67 (E), the GBC marker Ascl1 (F), the committed neuronal precursor marker NeuroD1 (G), the neuronal marker N-tubulin (H), and Sox2, which labels basal HBCs, proliferative GBCs, and apical sustentacular cells (D). Analysis of olfactory epithelium from the p63lox/lox conditional knockout reveals that HBCs expressing p63 are significantly reduced (A). HBC markers such as Krt14 and ICAM1 are strikingly absent from YFP-lineage-traced cells in the mutant epithelium (B and C), whereas these mutant YFP-labeled cells can give rise to differentiated cells in the neuronal and nonneuronal lineages (D–H). The absence of Krt14 and ICAM1 expression indicates a failure to maintain the undifferentiated HBC cell fate during methimazole-induced regeneration. HBCs reside directly adjacent to the basal lamina, indicated by the dashed line; apical and basal aspects of the epithelium are indicated in (A). Images were captured from the septum in the middle and ventral regions of the olfactory epithelium. Scale bar represents 50 μm. See also Figure S3.
Neuron  , DOI: ( /j.neuron )
Copyright © 2011 Elsevier Inc. Terms and Conditions

5. Figure 4
Defects in HBC Self-Renewal in the Conditional p63 Knockout Revealed by Metabolic Labeling of Dividing Cells
(A) Cre recombinase activity in Krt5-CreER(T2);Rosa26YFP mice in the p63+/ and p63lox/lox backgrounds was induced with tamoxifen at 3 weeks of age. The olfactory epithelium was injured with methimazole 36 hr following tamoxifen induction; 24 hr after methimazole treatment—a time at which HBCs start to proliferate in response to injury—animals were pulsed with the thymidine analog EdU to label dividing HBCs. Animals were fixed at 72 hr post-injury, and the position of YFP(+),EdU(+) double-labeled cells—which represent descendants of HBCs that had proliferated in response to injury—was assessed in control (p63+/) and mutant (p63lox/lox) mice.
(B) In controls, YFP(+),EdU(+) cells include cells directly apposed to the basement membrane (comprising regenerated HBCs; arrowheads) as well as globose suprabasal cells (representing differentiating cells).
(C) In the p63lox/lox mutants, fewer YFP(+),EdU(+) HBCs are observed, whereas YFP(+),EdU (+) suprabasal cells are still formed.
(D) Quantitation of the number of YFP(+),EdU(+) cells reveals a significant difference in the number of basal double-positive cells between control (n = 5) and mutant (n = 3; p = 0.014, unpaired two-tailed t test; error bars represent standard deviations) animals. By contrast, we observe no significant differences between the numbers of total EdU(+) cells, total YFP(+),EdU(+) cells, or suprabasal YFP(+),EdU(+) cells.
Dashed lines in (B) and (C) indicate the positions of the basal lamina; apical and basal aspects of the olfactory epithelium are indicated in (B). Images were captured from the septum in the middle and ventral regions of the olfactory epithelium. Scale bar represents 50 μm.
Neuron  , DOI: ( /j.neuron )
Copyright © 2011 Elsevier Inc. Terms and Conditions

6. Figure 5 Spontaneous Differentiation of HBCs in the Absence of p63
Uninjured olfactory epithelium from Krt5-CrePR;Rosa26YFP P12 mice was examined by immunohistochemistry for expression of the YFP-lineage tracer and cell-type-specific markers in the p63+/ and p63lox/lox backgrounds and visualized by confocal microscopy.
(A) Comparison between control (left) and mutant (right) shows the expected reduction in p63-positive, YFP-lineage-traced cells. Note the appearance of suprabasal YFP-labeled cells in the p63lox/lox conditional knockout, which are all but absent in the control background.
(B–H) Ablation of the p63 gene causes spontaneous differentiation and concomitant loss of HBCs. YFP-lineage-traced cells expressing the HBC markers Krt14 (B) and ICAM1 (C) are significantly reduced in the mutant. In the absence of p63, HBCs spontaneously differentiate, as evidenced by the expression of the proliferative marker Ki67 (E), the GBC marker Ascl1 (F), the committed neuronal precursor marker NeuroD1 (G), and the neuronal marker N-tubulin (H). Expression of Sox2, which labels both basal proliferative GBCs and apical sustentacular cells, localizes to YFP-lineage-traced cells in mutant tissue (D). Controls are either wild-type or heterozygous lox/+ at the p63 locus. HBCs reside directly adjacent to the basal lamina, indicated by the dashed line in each panel. Images were captured from the septum in the middle and ventral regions of the olfactory epithelium. Scale bar represents 50 μm. See also Figures S3 and S4.
(I–M) The percentages of YFP-labeled cells expressing specific markers were quantitated in uninjured olfactory epithelium at P12 in the p63+/+ and p63lox/lox backgrounds.
(I) Percentage of YFP-labeled cells expressing p63 is significantly reduced in the mutant compared to control (23% versus 97%).
(J) Percentage of YFP-labeled cells residing in suprabasal locations—reflecting differentiated cell types—is significantly higher in the mutant compared to wild-type epithelium (50% versus 0.15%).
(K and L) Percentages of lineage-traced cells comprising proliferative cells (labeled with Ki67) and GBC progenitors (labeled with Ascl1) are increased in the mutant relative to control (Ki67: 38% versus 9.7%; Ascl1: 12% versus 1.9%).
(M) Cell death as assayed by the detection of cleaved Caspase-3 is slightly, but not significantly, elevated in the mutant versus wild-type olfactory epithelium. Error bars represent standard deviations; ∗∗∗p < 0.002; ∗∗p < n.s. represents not significant (p = 0.36). n = 3 in p63+/+ mice; n = 4 in p63lox/lox mice for each marker.
Neuron  , DOI: ( /j.neuron )
Copyright © 2011 Elsevier Inc. Terms and Conditions